系统性红斑狼疮患者中抗中性粒细胞胞质抗体和细胞因子的关系

目的　探讨抗中性粒细胞胞质抗体 (ANCA)与系统性红斑狼疮 (SLE)血管炎临床特点的关系。检测SLE血管炎患者肿瘤坏死因子 (TNF) α、白细胞介素 (IL) 6等细胞因子的血清水平 ,探讨ANCA与SLE血管炎细胞因子的关系。方法　 6 0例SLE活动期患者和 30名正常对照组均通过间接免疫荧光法 (IIF)检测ANCA与酶联免疫吸附试验 (ELISA)检测抗髓过氧化物酶 (MPO)抗体 (即MPO ANCA) ,观察两组ANCA与抗MPO抗体的阳性率。通过ELISA法检测 30例SLE活动期患者和 30名健康对照者 ,外周血TNT α、白细胞介素 (IL) 6水平。结果　 6 0例SLE患者 1 5例ANCA阳性 ,均为核周型抗中性粒细胞胞质抗体 (pANCA) ,阳性率为 2 5 % ,抗MPO抗体 8例阳性(IIF法ANCA均阳性 ) ,阳性率为 1 3 3% ,健康对照组ANCA及抗MPO抗体均为阴性。病程超过 1年 ,伴有肾炎、浆膜炎、皮肤血管炎组 ,ANCA阳性率高 ,与无相应特点的对照组差异均有显著性 (P <0 0 5 ) ;有关节炎比无关节炎组ANCA阳性率高 (P =0 0 5 )。SLE活动期患者与对照组相比 :SLE组TNF α、IL 6水平较高 (P <0 0 5 )。抗MPO抗体阳性组与抗MPO抗体阴性组相比 :TNF α、IL 6水平升高 ,两组差异有显著性 (P <0 0 5 )。结论　部分SLE病人血清中可检测到 pANCA及抗MPO抗体 ,pANCA与SLE某些     

系统性血管炎抗内皮细胞抗体与抗中性粒细胞胞质抗体的相关性初探

目的以来源于大小血管内皮细胞的两种永生细胞株为底物,检测系统性血管炎血清中抗内皮细胞抗体(AECA),分析其与抗中性粒细胞胞质抗体(ANCA)的相关性。方法细胞酶联免疫吸附试验(Cyto-ELISA)法检测血清中AECA;间接免疫荧光法(IIF)及抗抗体结合内皮细胞表面的蛋白酶3(PR3)、抗MPO-ELISA检测血清中ANCA;IIF及抗PR3、抗MPO-ELISA检测细胞株中PR3及MPO;反转录—聚合酶链反应(RT-PCR)法检测细胞株中PR3及MPOmRNA。结果AECAEA(EA.hy926为底物所测AECA)阳性率33.6%(41/122),AECAHMEC(HMEC-1为底物所测AECA)37.7%(46/122),ANCA35.3%(43/122),AECAEA或AECAHMEC与ANCA串联诊断系统性血管炎敏感性分别为59.8%(73/122)或60.7%(74/122)。AECAEA与ANCA,AECAHMEC与ANCA分别行配对字2检验,差异均无显著性(P>0.05),符合率仅分别为49.2%及51.6%。EA.hy926和HMEC-1中蛋白水平及mRNA水平均无PR3和MPO的表达。结论EA.hy926与HMEC-1中无PR3、MPO蛋白水平的表达,ANCA与AECA可能是两种相互独立的抗体,串联检测可提高诊断敏感性。     

抗中性粒细胞胞质抗体的检出率及其靶抗原研究

目的　了解抗中性粒细胞胞质抗体 (ANCA)阳性检出率、流行病学特点及其靶抗原。方法　应用间接免疫荧光法 (IIF)、抗髓过氧化物酶 (MPO)和抗蛋白酶 3(PR3)酶联免疫吸附试验(ELISA)对近年送检的怀疑小血管炎的 5 6 0 4例患者血清进行了检测 ,对IIF ANCA阳性而抗MPO和抗PR3抗体均阴性的血清还进行了其他 5种ANCA特异性靶抗原的检测。并初步对ANCA阳性患者流行病学特点进行分析。结果　IIF ANCA检出率为 5 3% ,阳性检出最多在 7、8及 12月份。另外所有血清中有 390例 (7% )ANA阳性。所有血清进行抗MPO和抗PR3 ELISA检测 ,抗MPO抗体阳性 2 13例 ,抗PR3抗体阳性 32例 ,两者同时阳性 5例。 4 8例不识别MPO和PR3而IIF法阳性的血清中 13例识别其他已知靶抗原 ,识别杀菌 /通透性增高蛋白 (BPI)、人弹力蛋白酶 (HLE)、组蛋白酶G (CG)、天青杀素 (AZU)和乳铁蛋白 (LF)等靶抗原的血清分别为 7、5、1、1、0例 ,其中 1例为抗BPI和抗HLE ANCA同时阳性。 85 %的IIF ANCA阳性患者确诊为ANCA相关小血管炎。这些患者中 ,抗MPO和抗PR3的比例约为 7∶1:男女比例为 1∶1 12 ,年龄 7～ 79岁 ,平均 5 3 1岁 ,>6 0岁的老年人男女比例为 1 17∶1,而年龄 <2 0岁患者男女比例为 1∶4。结论　ANCA相关疾病在我国并不少见 ,以IIF法检     

抗中性粒细胞胞浆抗体在肾炎综合征中的检测及其临床意义

目的　探讨检测抗中性粒细胞胞浆抗体 (ANCA)及其靶抗原在肾炎综合征中的临床意义。方法　应用间接免疫荧光 (IIF)法检测 10 0例肾炎综合征患者血清抗中性粒细胞胞浆抗体 ,对其阳性的 2 9例用酶联免疫吸附试验 (ELISA )检测靶抗原髓过氧化物酶 (MPO)和蛋白酶 3(PR3 )。结果　IIF检测肾炎综合征ANCA阳性率为 2 9% ,其中胞浆型 10 %、核周型 19%。急进型肾炎、狼疮性肾炎、紫癜性肾炎阳性率分别为 5 6%、2 0 %和 15 %。ELISA急进性肾炎和紫癜性肾炎大多数识别靶抗原MPO ,狼疮性肾炎ANCA不识别MPO或PR3。结论　ANCA在急进性肾炎和狼疮性肾炎中阳性率较高 ,检测ANCA对判断狼疮性肾炎活动及疗效具有参考价值     

抗蛋白酶-3抗体在韦格纳肉芽肿病和其他血管炎中的临床应用研究

目的探讨抗蛋白酶-3(PR3)抗体在韦格纳肉芽肿病(WG)和其他血管炎患者中的表达和临床意义。方法选取2001年3月至2006年7月山西医科大学第二医院确诊的住院患者576例。系统性血管炎组111例,其中9例WG(包含21份跟踪随访血清)；结缔组织病(CTD)组403例；各型肾小球疾病患者62例及健康对照30名,均采用酶联免疫吸附试验(ELISA)检测抗PR3抗体、抗髓过氧化物酶(MPO)抗体；采用间接免疫荧光法(IIF)检测抗中性粒细胞胞质抗体(ANCA),观察抗PR3与ANCA在WG和其他血管炎中的阳性率,且追踪WG治疗前后抗PR3吸光度值和ANCA的滴度变化。结果588例血清中抗PR3抗体阳性23例,分别为WG 15例(15/21,71．4％)；系统性红斑狼疮(SLE)6例(6/213,2．8％)：类风湿关节炎(RA)1例(1/135,0．7％)；混合结缔组织病(MCTD)1例。大动脉炎、白塞病,过敏性紫癜等常见的原发性系统性血管炎、肾病组、健康对照组未发现抗PR3抗体阳性。抗PR3抗体和胞质型ANCA(cANCA)在WG中阳性率最高,差异有统计学意义(P＜0．05)。抗PR3对WG诊断的敏感性71．42％,特异性98．58％。联合应用抗PR3与cANCA诊断WG的敏感性61．90％,特异性99．82％。抗PR3吸光度值、ANCA的滴度及伯明翰血管活动度评分(BVAS)随治疗好转下降。结论抗PR3抗体是诊断WG的一种敏感、特异标记抗体,抗PR3抗体和ANCA同时在临床应用,有利于WC和其他系统性血管炎的早期诊断和鉴别诊断。抗PR3抗体还可作为临床疗效观察指标。     

抗中性粒细胞胞浆抗体检测的临床应用

抗中性粒细胞胞浆抗体 (antineutrophilcyto plasmicantibodies ,ANCA )是一种以中性粒细胞胞质成分为靶抗原的自身抗体。 1982年 ,Davies首次在节段坏死性肾小球肾炎的患者中描述 ,并认为ANCA与罗斯河病毒感染有关。直到 1985年 ,VanderWoude才将ANCA和韦格纳肉芽肿 (WG)联系起来 ,以后Savage等发现显微镜下多血管炎 (MPA)中也存在ANCA ,且与病情活动密切相关。在当今血管炎的诊断和分类中ANCA的作用与地位已愈来愈重要。血管炎患者体内 ,存在两种靶抗原即蛋白酶3(PR3)和髓过氧化酶 (MPO)。他们主要存在于嗜中性粒细胞嗜天青…     

65例系统性红斑狼疮患者抗中性粒细胞胞浆抗体的检测分析

目的探讨抗中性粒细胞胞浆抗体(ANCA)与系统性红斑狼疮(SLE)的关系及临床意义.方法回顾性分析65例SLE患者的ANCA检测结果; ANCA与SLE主要临床表现、实验室检查结果的关系; SLE患者病情活动组与非活动组ANCA阳性率的比较.结果 IIF法检测ANCA在SLE中的阳性率是61.5 %,ANCA阳性组中有血管炎皮损(67.5 %)及浆膜炎(55.0 %)者明显高于阴性组(P<0.05);同时ANCA阳性组与阴性组比较,在24 h尿蛋白大于0.5 g/L,血红蛋白低于90 g/L,抗ds-DNA抗体阳性,低补体血症方面差别有统计学意义(P<0.05).结论提示ANCA可能是判断SLE病情复发与缓解的一个有用指标,推测ANCA与活动性狼疮肾炎有关.     

抗中性粒细胞胞质抗体与系统性红斑狼疮的相关性研究

目的探讨抗中性粒细胞胞质抗体（ANCA）与系统性红斑狼疮（SLE）的相关性及其临床意义。方法收集活动组和非活动组各50例SLE患者的临床和实验室资料,间接免疫荧光（IIF）方法检测患者血清ANCA,AN-CA阳性者加做髓过氧物酶（MPO）、蛋白酶3（PR3）的酶联免疫检测（ELISA）。结果①活动组SLE患者ANCA阳性率（64.0%）高于非活动组（20.0%）及对照组（2.0%）（P均〈0.05）;②ANCA阳性组与阴性组比较,在临床表现（胸膜炎、心包炎、肾损害方面）2、4 h尿蛋白〉0.5 g/d、抗ds-DNA抗体阳性、低补体血症方面差异有统计学意义（P〈0.05）;③ANCA阳性的狼疮肾炎组MPO-ANCA阳性率（71.4%）高于非狼疮肾炎组（25.0%）,P=0.038。结论 ANCA与SLE发病和疾病活动有关,可能是判断SLE病情复发与缓解的一个有用指标;MPO-ANCA可能和狼疮肾炎存在密切相关性。     

426例抗中性粒细胞胞浆抗体相关性小血管炎患者多系统临床表现和肾脏病理分析

目的 分析426例抗中性粒细胞胞浆抗体（ANCA）相关性小血管炎患者多系统的临床和病理表现。方法 回顾性分析我院1997年-2004年6月检测并明确诊断的426例ANCA相关性小血管炎患者的临床病理资料。结果 426例患者中,70例胞浆型ANCA（cANCA）阳性,均识别蛋白酶3（PR3）;354例环核型ANCA（pANCA）阳性,均识别髓过氧化物酶（MPO）。201例（47．2％,201／426）患者是在发病后3个月内确诊。临床表现呈多器官受累,其中cANCA阳性者皮疹、关节痛、眼、鼻受累的发生率显著高于pANCA阳性者,而pANCA阳性者。肾脏受累和乏力的发生率显著高于cANCA阳性者。多数患者有贫血,血沉增快,C反应蛋白增高。采用糖皮质激素联合环磷酰胺进行强化免疫抑制治疗,诱导缓解期的缓解率为88．5％。结论 ANCA相关性小血管炎在我国并非少见,临床表现呈多器官受累,ANCA检测有助于早期诊断。     

丙基硫氧嘧啶诱发抗中性粒细胞胞质抗体阳性小血管炎三例并文献复习

目的研究丙基硫氧嘧啶(PTU)诱发的抗中性粒细胞胞质抗体(ANCA)阳性小血管炎的临床病理表现。方法对我院近2年诊治的3例FPU引起的ANCA阳性小血管炎进行临床病理分析并复习相关文献。结果3例病人均为女性．年龄24～32岁．平均28．3岁。服PTU时间2～17年．3例均出现皮疹、关节痛．但不累及肾、脑、肺、造血等重要脏器，均为核周型ANCA(pANCA)．髓过氧化物酶ANCA(MPO-ANCA)显著增高(90～260EU／ml)，1例并胞质型ANCA．2例抗甲状腺球蛋白抗体升高，1例抗核抗体(ANA)阳性，而同期服DTU、他巴唑(MMI)无小血管炎临床表现的格雷夫斯病血清14份、31份pANCA均阴性，PTU组有2例MPO-ANCA低水平增高(20～26EU／ml)。3例患者均立即停用PTU，改服MMI、甲亢平各1例，同时应用糖皮质激素，临床症状均缓解，各种自身抗体滴度降低。结论PTU可诱发ANCA阳性小血管炎，应及时发现停药，糖皮质激素治疗有效。     

抗日本血吸虫抗独特型抗体的制备及初步鉴定和应用

本研究用1株针对日本血吸虫尾蚴和童虫表膜抗原的单克隆抗体(N15D9)免疫新西兰兔制备多克隆抗N15D9独特型抗体。结果表明,多克隆抗N15D9独特型抗体具有较高的抗体滴度,经用间接免疫荧光法和ELISA分析,该抗体显示较高的结合特异性,与具有相同靶抗原结合特异性的单克隆抗体有中度的交叉反应。当用抗N15D9独特型抗体作为诊断抗原检测日本血吸虫感染的病人血清时,其阳性检出率为39％。本研究结果初步揭示,抗N15D9独特型抗体具有一定的分子模拟作帮和诊断应用的潜能。     

The influence of anti-endothelial/antiphospholipid antibodies on fibrin formation and lysis on endothelial cells

The prothrombotic mechanisms associated with antiphospholipid antibodies remain incompletely defined. Antibody binding to endothelial cells in vitro is a feature of antiphospholipid antibody-positive sera. We hypothesised that impairment of endothelium-dependent fibrinolysis by antiphospholipid/anti-endothelial antibodies is a contributory factor in the pathogenesis of thrombosis. We also aimed to confirm the displacement of annexin-V from endothelial cells and enhanced fibrin formation. Binding of immunoglobulin (Ig) from antiphospholipid antibody-positive sera to endothelial cells was examined using a cell-based enzyme-linked immunosorbent assay. Effects on fibrin formation and lysis were examined on cultured endothelial cell monolayers. Plasminogen activator inhibitor-1 (PAI-1) was assayed in supernatants. We confirmed antibody binding to endothelial cells. With four of 14 antiphospholipid antibody-positive sera there was some prolongation of fibrin clot lysis time, consistent with impairment of endothelial fibrinolytic activity. Secretion of PAI-1 was significantly correlated with clot lysis time on endothelial cell monolayers incubated with antiphospholipid/anti-endothelial antibody-positive sera, but not with control sera. IgG from antiphospholipid antibody-positive sera had little effect on endothelial cell surface annexin-V expression. We conclude that impaired endothelial fibrinolysis is a potential prothrombotic mechanism in subjects with antiphospholipid antibodies. We were unable to confirm enhanced displacement of annexin-V from endothelium by antiphospholipid antibodies.     

输血前11606例患者HIV和HCV及TP抗体检测结果分析

目的为避免因输血传播疾病引起医疗纠纷和防止职业感染。方法采用酶联免疫吸附试验对2010-01～2011-03 11 606例就医患者进行输血前人类免疫缺陷病毒(HIV)抗体、丙型肝炎病毒(HCV)抗体、梅毒螺旋体(TP)抗体三项指标筛查。结果共检出阳性例数856例。结论患者输血前进行三项感染性指标检测,对诊断和预防医患交叉感染、减少医疗纠纷的发生具有重要意义。     

Changes in titers of antimitochondrial and antinuclear antibodies during the course of primary biliary cirrhosis

A case of primary biliary cirrhosis (PBC) in whom a complete biochemical (serum bilirubin, transaminases and alkaline phosphatase) remission was noted after combination treatment with ursodeoxycholic acid (UDCA) and corticosteroid is reported. The antimitochondrial antibody (AMA) detected by indirect immunofluorescence was initially positive, and the antinuclear antibody (ANA) was negative, but these two antibodies subsequently fluctuated independently (AMA-positive/ANA-negative, AMA-negative/ANA-negative, AMA-negative/ANA-positive, AMA-positive/ANA-positive, and again AMA-negative/ANA-positive) in spite of a lack of histopathological improvement in the liver after treatment. The clinical presentation in our case suggests that in some cases the diagnosis of PBC or so-called autoimmune cholangitis (AIC) might depend on the 'phase' of the same disease. Our results also suggest that detailed immunoreactive profiles against 2-oxo-acid dehydrogenase complex (2-OADC) enzymes by using immunoblotting, together with a serial histological examination, should provide more precise information for a diagnosis of PBC.     

酶联免疫吸附试验检测弓形虫体IgA抗体及其临床应用的初步研究

试用弓形虫膜抗原ELISA对先天性弓形虫感染的畸形围产儿和后天获得性弓形虫感染者进行了特导性IgA抗体检测。11例先天性弓形虫宫内感染畸形围产儿的15份标本,6份检出IgA抗体,7份同时检出IgA;IgM抗体,两份仅检出IgM抗体。8份弓形虫抗体阳性的孕妇血清皆检出较强的IgA抗体。后天获得性弓形虫近期感染者32份血清,IgA抗体阳性3份,IgA、IgM抗体阳性8份,IgM抗体阳性21份;30份慢性感染者血清未有测到IgA抗体。对照组30份抗体阴性的孕妇血清及其围产儿的脐血检测结果抗体皆为阴性。IgA抗体是弓形虫感染早期的一个重要标志物,与IgM、IgG抗体同时检测,对临床急性弓形虫感染的诊断和疗效观察有着极其重大意义。     

免疫人群乙肝抗体阳性率调查及乙肝核心抗体检测方法探讨

目的 调查我院检测人群乙型肝炎病毒表面抗体(HBsAb)及乙型肝炎病毒核心抗体(HBcAb)阳性率,并探讨检测方法选择及检测操作模式对免疫人群乙肝核心抗体结果的影响。方法 统计2012—2013年用化学发光微粒子免疫检测法(CMIA)和酶联免疫竞争抑制一步法(ELISA)检测乙肝人群的HBsAb及HBcAb阳性率,并按≤2岁、2~20岁、>20岁3个年龄分段比较;收集92例免疫人群样本用CMIA和3种ELISA法检测HBcAb;用同种ELISA法试剂,改变操作模式进行HBcAb检测。结果 3组年龄段检测人群HBsAb两种检测方法统计的阳性率无统计学差异,≤2岁组阳性率最高;HBcAb用CMIA法统计的阳性率在≤2岁组和2~20岁组的免疫人群组低于ELISA法。 92例样本用CMIA法检测HBcAb阳性率为2.2%;用3种ELISA法试剂检测阳性率分别为79.3%、82.6%及94.6%;3种ELISA法试剂检测阳性率无统计学差异。92例样本改变操作模式,用同种ELISA法检测试剂检测结果有19例为阴性,差异有统计学意义。结论 检测人群HBsAb检出率在2岁及以下人群最高,随年龄增长抗体下降。 HBcAb用CMIA法检测在免疫人群中检出阳性率明显低于EIISA法。ELISA竞争抑制一步法检测HBcAb易造成假阳性,如改用HBcAb单项加样操作或选用化学发光微粒子免疫检测法(CMIA)检测,可明显减少假阳性。     

Determination of antiplatelet antibody activity in sera cytotoxic for human lymphocytes

Pooled serum aliquots obtained from sensitized potential renal allograft recipients on chronic hemodialysis were evaluated for their lymphocytotoxicity titers against the lymphocytes and then for alloantibodies against the platelets of 7 random donors by 5 methods. Platelet donor specific lymphocytotoxicity was present in 93% of 42 combinations. Of the positive combinations, 57% had a positive test for antiplatelet activity by the 14C serotonin release assay, 16% by the platelet aggregation method, and 19% as judged by acid phosphatase availability on the platelet membrane. No serum tested released beta-glucoronidase or lactic dehydrogenase. No correlation of the height of the titer of antiplatelet activity with that for lymphocytoxicity was detected. Thus, even in sera demonstrating significant activity against donor lymphocyte antigens, detection of associated platelet antibody activity is not uniform. Thus, a positive lymphocytoxic titer does not necessarily predict detectable antiplatelet activity. Therefore, additional tests for detection of antiplatelet activity should also be considered. This study shows that of the tests evaluated, the 14C serotonin release assay is the most sensitive for detection of antiplatelet antibodies.     

Pentameric complex of viral glycoprotein H is the primary target for potent neutralization by a human cytomegalovirus vaccine

Human cytomegalovirus (HCMV) can cause serious morbidity/mortality in transplant patients, and congenital HCMV infection can lead to birth defects. Developing an effective HCMV vaccine is a high medical priority. One of the challenges to the efforts has been our limited understanding of the viral antigens important for protective antibodies. Receptor-mediated viral entry to endothelial/epithelial cells requires a glycoprotein H (gH) complex comprising five viral proteins (gH, gL, UL128, UL130, and UL131). This gH complex is notably missing from HCMV laboratory strains as well as HCMV vaccines previously evaluated in the clinic. To support a unique vaccine concept based on the pentameric gH complex, we established a panel of 45 monoclonal antibodies (mAbs) from a rabbit immunized with an experimental vaccine virus in which the expression of the pentameric gH complex was restored. Over one-half (25 of 45) of the mAbs have neutralizing activity. Interestingly, affinity for an antibody to bind virions was not correlated with its ability to neutralize the virus. Genetic analysis of the 45 mAbs based on their heavy- and light-chain sequences identified at least 26 B-cell linage groups characterized by distinct binding or neutralizing properties. Moreover, neutralizing antibodies possessed longer complementarity-determining region 3 for both heavy and light chains than those with no neutralizing activity. Importantly, potent neutralizing mAbs reacted to the pentameric gH complex but not to gB. Thus, the pentameric gH complex is the primary target for antiviral antibodies by vaccination.Human cytomegalovirus (HCMV) is an important pathogen in transplant patients (1–5), and its infection can lead to invasive end-organ diseases, such as pneumonitis and hepatitis, as well as vascular pathology contributing to graft failure (4, 6, 7). HCMV is also the most common cause of in utero viral infections in North America and Europe, affecting 0.5–2% of newborns annually (8–10). Congenital HCMV infection can lead to symptomatic diseases at birth and also cause developmental disabilities in children (10, 11). Maternal seropositivity before conception protects against congenital transmission (12, 13), and both maternal humoral and cellular immunity are likely to contribute to the protection (14–16). Antibodies in particular are important for preventing congenital infection, serving as the first line of defense against maternal infection. It may also play a role in preventing transmission to the fetus, supported by the results of a small, nonrandomized study in pregnant women with primary HCMV infection, in which the passive immunity of monthly infusions of HCMV hyperimmune human IgG (HCMV-HIG) (200 mg/kg maternal weight) was ∼60% effective in protecting against congenital HCMV infection (17, 18). These studies suggest that it is feasible to develop a vaccine for preventing congenital HCMV infection and its sequelae. However, despite the fact that the Institute of Medicine has identified development of an effective vaccine for prevention of congenital HCMV as a top priority since 1999 (19), progress toward this goal has only been incremental (8, 20, 21). One of the hurdles to the efforts is our limited understanding of component of natural immunity associated with protection against HCMV infection.HCMV is a large, complex virus, with a genome capable of encoding >150 proteins (22–26). Because of the strict species specificity, options of animal models for HCMV research are limited (27). Thus, the functions of most HCMV antigens in viral infection in vivo and their roles as targets for host immunity are poorly understood. Furthermore, culture systems of single cell types have limitations for studying HCMV pathogenesis. Immunohistochemistry studies showed that HCMV can infect varieties of cells in vivo, including endothelial, epithelial cells, fibroblasts, and leukocytes (28–36). Many HCMV end-organ diseases, such as pneumonitis and gastroenteritis, are due to infection of the epithelial/endothelial cells in the affected organ (35–39). However, common laboratory strains, such as AD169 and Towne, were culture-adapted in fibroblast cells, with genomic mutations (22, 24, 40) and, more importantly, have lost their tropism to endothelial and epithelial cells, in contrast to pathogenic clinical isolates (32, 33, 41, 42).Loss of viral tropism to endothelial and epithelial cells was mapped to various mutations in the viral UL131-128 locus, and these mutations abrogated the expression of the pentameric glycoprotein H (gH) complex, composed of gH, gL, UL128, UL130, and UL131 proteins, a determinant for viral tropism to endothelial and epithelial cells (42–44). Because the pentameric gH complex is missing in common laboratory strains (42, 43), its importance in viral tropism, viral pathogenesis, and vaccine design was not fully appreciated until recently (42, 45). With this understanding, it is not surprising that Towne virus and recombinant glycoprotein B (gB) vaccines, although with ∼50% efficacy against primary infection in the clinic (46–49), induced poor neutralizing titers against viral infection of epithelial cells, in contrast to immune sera from HCMV-seropositive donors (50, 51). Thus, missing the pentameric gH complex is likely a deficiency in antigen composition for both vaccines (50). Studies of monoclonal antibodies (mAbs) isolated from HCMV-seropositive donors or polyclonal IgG enriched for antigen specificity supported the hypothesis that the pentameric gH complex, not gB, appears to be important for neutralizing activity in human subjects with natural infection (52).We recently described an experimental vaccine virus in which expression of the pentameric gH complex was restored (53). Unlike the parental AD169 virus and the recombinant gB vaccine, this virus can elicit high levels of neutralizing antibodies in rabbits and rhesus macaques (53). To support clinical development of this vaccine centered its concept on the pentameric gH complex, we established a comprehensive panel of 45 mAbs from a single rabbit that received vaccination. Of the 45 mAbs, 25 had neutralizing activity against viral entry in epithelial cells, including 11 elite neutralizers with ≥10-fold greater potency than HCMV-HIG. Biochemical analysis demonstrated that all elite neutralizers preferentially bound to the virus expressing the pentameric gH complex, and the majority of elite neutralizers (8 of 11) specifically recognized a recombinant form of the pentameric gH complex. Interestingly, binding affinity for intact virions was not correlated with neutralizing activity. Moreover, genetic analysis of the 45 mAbs based on their heavy- and light-chain sequences identified at least 26 B-cell linage groups characterized by distinct binding or neutralizing properties. In addition, neutralizing antibodies had longer complementarity-determining region 3 (CDR3) for both heavy and light chains than those of antibodies with no neutralizing activity. These data establish the importance of the pentameric gH complex as the primary target for potent neutralizing antibodies by vaccination, and support development of an experimental HCMV vaccine featuring the pentameric gH complex.     

全人源肝癌噬菌体单链抗体基因的克隆及活性分析

从噬菌体单链抗体库中筛选克隆全人源肝癌抗体基因并进行活性鉴定.PCR鉴定阳性重组菌中人肝癌ScFv的插入率,以肝癌细胞SMMC-7721为抗原对所建抗体库进行4轮"吸附-洗脱-扩增"的亲和筛选.将筛选后的ScFv采用ELISA法鉴定其与人肝癌细胞的结合活性.ScFv基因插入率为70%.在亲和筛选过程中,肝癌噬菌体单链抗体得到富集,收获率逐轮提高,第4轮为第一轮的381倍.利用噬菌体抗体库技术筛选出了肝癌噬菌体单链抗体,且筛选后的抗体片段与人肝癌细胞有特异性的结合活性.